U.S. patent number 7,959,952 [Application Number 11/469,659] was granted by the patent office on 2011-06-14 for method for skin care.
This patent grant is currently assigned to Nuliv Holding Inc.. Invention is credited to Tsu-Chung Chang, Wen-Liang Chang, Hang-Ching Lin, Yuan Yang.
United States Patent |
7,959,952 |
Chang , et al. |
June 14, 2011 |
Method for skin care
Abstract
A method for skin care includes administering the water
insoluble fraction of Astragalus membranaceus, or astragaloside
compounds thereof to a subject in need thereof at an effective
amount for improving skin texture, reducing wrinkles, UV
protection, and/or providing an anti-aging effect.
Inventors: |
Chang; Wen-Liang (Taipei,
TW), Chang; Tsu-Chung (Taipei, TW), Lin;
Hang-Ching (Taipei, TW), Yang; Yuan (Taipei,
TW) |
Assignee: |
Nuliv Holding Inc. (Taipei,
TW)
|
Family
ID: |
39136928 |
Appl.
No.: |
11/469,659 |
Filed: |
September 1, 2006 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20080057010 A1 |
Mar 6, 2008 |
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Current U.S.
Class: |
424/725; 424/773;
424/74; 424/757 |
Current CPC
Class: |
A61K
8/9789 (20170801); A61Q 19/08 (20130101); A61K
31/56 (20130101); A61K 36/481 (20130101); A61K
31/704 (20130101); A61K 8/63 (20130101); A61K
2800/92 (20130101) |
Current International
Class: |
A61K
36/00 (20060101); A61K 8/97 (20060101); A61K
36/481 (20060101); A61K 36/48 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1318381 |
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Oct 2001 |
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CN |
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62012791 |
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Jan 1987 |
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JP |
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03220128 |
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Sep 1991 |
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JP |
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2005194246 |
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Jul 2005 |
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JP |
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2005073918 |
|
Jul 2005 |
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KR |
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2005/000248 |
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Jan 2005 |
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WO |
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Other References
Ma et al., Chemical analysis of radix Astragali (Huangqi) in China:
a comparison with its adulterants and seasonal variations. J Agric
Food Chem 50: 4861-4866, 2002. cited by examiner .
Ma et al., Chemical analysis of radix Astragalli (Huangqi) in
China: a comparison with its adulterants and seasonal variations. J
Agric Food Chem 50: 4861-4866, 2002. cited by examiner .
International Search Report for the corresponding International
Patent Application No. PCT/US07/77326; Mailed Apr. 11, 2008; 3
pages. cited by other .
Viennet, et al., "Effects of astragaloside on growth and apoptosis
of fibroblasts from human skin with and without wrinkle in vitro",
Chinese Journal of Medical Aesthetics and Cosmetology, vol. 12,,
No. 2, p. 93-97, 2006. cited by other.
|
Primary Examiner: Mi; Qiuwen
Attorney, Agent or Firm: Occhiuti Rohlicek & Tsao
LLP
Claims
We claim:
1. A method for enhancing collagen expression in skin cells,
comprising topically administering a composition, including a water
insoluble fraction of Astragalus membranaceus as the active
ingredient, to a skin area in a subject in an amount effective for
enhancing collagen expression, wherein the water insoluble fraction
of Astragalus membranaceus is prepared by a process including:
extracting Astragalus membranaceus with alcohol to obtain an
alcohol extract; dispersing the alcohol extract, in dry form, in
water and n-butanol at a volume ratio of 1:1; and collecting the
water insoluble fraction.
2. The method according to claim 1, wherein the composition is a
cosmetic cream.
3. The method of claim 1, wherein the composition further comprises
at least one cosmetically acceptable carrier.
4. The method of claim 1, wherein the composition is administered
to a healthy subject.
5. The method of claim 1, wherein the skin area is a facial skin
area.
6. The method of claim 4, wherein the skin area is a facial skin
area.
Description
BACKGROUND OF THE INVENTION
The present invention relates to a method for skin care, and
particularly to a method for skin care using the water insoluble
fraction, organic fraction, or active components of Astragalus
membranaceus.
Astragalus is derived from the root of a plant, known as Astragalus
membranaceus. It is also known as milk vetch root (referring to
astragalus species that grow in the United States) and Huang-Qi.
Astragalus has often been used by practitioners of traditional
Chinese medicine to strengthen or tone the body's overall vitality,
improve digestion, and support the spleen. Studies confirmed that
it contains medicinally active compounds, including a
polysaccharide that stimulates the immune system.
Research has also shown that subjects with advanced cancer showed a
two to three fold increase in the strength of their immune response
after being given astragalus. A second study showed that astragalus
boosted immune response, even in animals that were treated with an
immunosuppressive drug, cyclophosphamide. Astragalus is taken in
China by cancer patients to boost immunity after drug or radiation
treatment. It may protect body cells against heavy metals and
chemical toxins.
However, none of the studies is directed to an extract from
Astragalus membranaceus used for skin care.
BRIEF SUMMARY OF THE INVENTION
One aspect of the invention is to provide a method for skin care,
which comprises administering the water insoluble fraction of
Astragalus membranaceus to a subject in need thereof at an
effective amount for improving skin texture, reducing wrinkles,
ultraviolet (UV) protection, and anti-aging effects. In an example
of the invention, the water insoluble fraction of Astragalus
membranaceus is prepared by a process comprising the steps of
extracting Astragalus membranaceus with an alcohol to obtain an
alcohol extract; and extracting the alcohol extract with water
washed to obtain a water insoluble fraction.
In another aspect, the invention provides a method for skin care
comprising administering a cycloartane compound of formula (I) to a
subject in need thereof at an effective amount for anti-aging
effect and UV protection:
##STR00001## wherein R.sub.1 is selected from a group consisting of
H, OH, O-acetyl, O-xylopyranosyl, O-(2-acetylxylopyranosyl),
O-(3-acetylxylopyranosyl),
O-(2,3-diacetylxylopyranosyl),O-(2,4-diacetylxylopyranosyl),
O-xylopyranosyl-(1-2)-.beta.-D-glucopyranosyl,
O-xylopyranosyl-(1-2)-.alpha.-arabinopyranosyl, R.sub.2 is selected
from a group consisting of H, OH, O-acetyl, O-glucopyranosyl,
O-xylopyranosyl, R.sub.3 is selected from a group consisting of H,
OH and O-acetyl, and R.sub.4 is selected from a group consisting
of
##STR00002##
Additional features and advantages of the present invention will be
set forth in part in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the invention. The features and advantages of the invention will
be realized and attained by means of the elements and combinations
as described.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings.
In the drawings:
FIG. 1 is a SDS-PAGE gel image showing suppression of MMP-1
expression in human HDF cells treated with astragalosides.
DETAILED DESCRIPTION OF THE INVENTION
Reference will now be made in detail to the present embodiments of
the invention. For a better understanding of the present invention,
some of the terms used herein are explained in more detail.
As used herein, the article "a" or "an" means one or more than one
(that is, at least one) of the grammatical object of the article,
unless otherwise made clear in the specific use of the article in
only a singular sense.
An "effective amount" as used herein refers to an amount that
provides anti-aging effect, ultraviolet (UV) protection or both in
a subject in need thereof. For those skilled in the art, the
effective amount, as well as dosage and frequency of
administration, may easily be determined according to their
knowledge and standard methodology of merely routine
experimentation based on the present disclosure.
The present invention provides a method for skin care, which
comprises administering a water insoluble fraction of Astragalus
membranaceus to a subject in need thereof at an effective amount
for improving skin texture, reducing wrinkles, UV protection and
anti-aging effect. In an example of the invention, the water
insoluble fraction of Astragalus membranaceus is prepared by a
process comprising the steps of extracting Astragalus membranaceus
with an alcohol to obtain an alcohol extract; and extracting the
alcohol extract with water to obtain a water insoluble
fraction.
In accordance with one embodiment, the water insoluble fraction of
Astragalus membranaceus comprises astragaloside of the formula
(I):
##STR00003## wherein R.sub.1 is selected from a group consisting of
H, OH, O-acetyl, O-xylopyranosyl, O-(2-acetylxylopyranosyl),
O-(3-acetylxylopyranosyl), O-(2,3-diacetylxylopyranosyl),
O-(2,4-diacetylxylopyranosyl),
O-xylopyranosyl-(1-2)-.beta.-D-glucopyranosyl,
O-xylopyranosyl-(1-2)-.alpha.-arabinopyranosyl, R.sub.2 is selected
from a group consisting of H, OH, O-acetyl, O-glucopyranosyl,
O-xylopyranosyl, R.sub.3 is selected from a group consisting of H,
OH and O-acetyl, and R.sub.4 is selected from a group consisting
of.
##STR00004##
In a example of the invention, the water insoluble fraction
comprises astragaloside of the formula (II):
##STR00005## wherein R.sub.A, R.sub.B, R.sub.C, and R.sub.D are
selected from a group consisting of acetyl (Ac), hydrogen (H) and
glucopyranoside (Glc).
In a preferable embodiment of the invention, the water insoluble
fraction of Astragalus membranaceus comprises astragaloside of the
formula (II), wherein R.sub.A is Ac, R.sub.B is Ac, R.sub.C is H
and R.sub.D is Glc; for example, astragaloside I.
In accordance with another preferable embodiment of the invention,
the water insoluble fraction of Astragalus membranaceus comprises
astragaloside of the formula (II), wherein R.sub.A is Ac, R.sub.B
is H, R.sub.C is H and R.sub.D is Glc; for example, astragaloside
II.
In accordance with a preferable embodiment of the invention, the
water insoluble fraction of Astragalus membranaceus comprises
astragaloside of the formula (II), wherein R.sub.A is Glc, R.sub.B
is H, R.sub.C is H and R.sub.D is H; for example, astragaloside
III.
In accordance with a preferable embodiment of the invention, the
water insoluble fraction of Astragalus membranaceus comprises
astragaloside of the formula (II), wherein R.sub.A is H, R.sub.B is
H, R.sub.C is H and R.sub.D is Glc; for example, astragaloside
IV.
In accordance with one preferable embodiment of the invention, the
water insoluble fraction of Astragalus membranaceus comprises
astragaloside of the formula (II), wherein R.sub.A is Glc, R.sub.B
is H, R.sub.C is H and R.sub.D is Glc; for example, astragaloside
VI.
In accordance with a preferable embodiment of the invention, the
water insoluble fraction of Astragalus membranaceus comprises
astragaloside of the formula (II), wherein R.sub.A is Ac, R.sub.B
is H, R.sub.C is Ac and R.sub.D is Glc; isoastragaloside I.
In accordance with a preferable embodiment of the invention, the
water insoluble fraction of Astragalus membranaceus comprises
astragaloside of the formula (II), wherein R.sub.A is H, R.sub.B is
Ac, R.sub.C is H and R.sub.D is Glc; isoastragaloside II.
In another example of the invention, the water insoluble fraction
of Astragalus membranaceus comprises astragaloside of formula (I),
wherein R.sub.1 is OH, R.sub.2 is O-glucopyranosyl, R.sub.3 is OH,
and R.sub.4 is
##STR00006## For example, the water insoluble fraction comprises
cylcoastragenol-6-O-.beta.-D-glucopyranoside.
As the results of the experiments performed in the invention, the
water insoluble fraction was administered to enhance cellular
expression and secretion of collagens I and III, promote
glucosamine and proline uptakes, up-regulate the hyaluronic acid
(HA) expression, and down-regulate the expression and proteolytic
activity of matrix metalloproteinases (MMPs). In an embodiment of
the invention, the cells were treated with a dosage of about
0.25-40 .mu.g/mL of the water insoluble fraction to obtain a good
performance as described above.
In addition, the invention provides a method for skin care
comprising administering an astragaloside compound of formula (I)
to a subject in need thereof at an effective amount for providing
anti-aging effect and UV protection. The definitions of the
substituents are defined as above.
In an embodiment of the invention, the astragaloside compound
comprises astragaloside of the formula (II), the definitions of the
substituents and the preferable embodiments are given above.
In an embodiment of the invention, the astragaloside compound
comprises astragaloside of formula (I), wherein R.sub.1 is OH,
R.sub.2 is O-glucopyranosyl, R.sub.3 is OH, and R.sub.4 is
##STR00007## for example,
cylcoastragenol-6-O-.beta.-D-glucopyranoside.
Similarly, as the results of the experiments conducted in the
invention, the cellular expression of collagens I and III was
enhanced, glucosamine and proline uptakes were promoted, HA
expression was up-regulated, and the expression and proteolytic
activity of MMP were down-regulated when an astragaloside compound
according to the invention was administered, at such as a dosage of
about 0.01-10 .mu.M. The enhanced MMP expression triggered by UV
irradiation was also suppressed, suggesting that this compound
protect collagen from degradation elicited by MMP. The
astragalosides of formulae (I) and (II) and the water insoluble
fraction can serve as agents for the prevention and treatment of
skin aging and UV protection for the skin.
In another embodiment of the invention, when the cosmetic cream
comprising the water insoluble fraction of Astragalus membranaceus
was applied on test subjects' skin, the facial skin of the test
subjects looked more radiant and whitening as indicated by
parameters for measuring the erythema and melanin. Also, the pH of
the facial skin was reduced to a level, which prevented growth of
the microorganism. Furthermore, the wrinkle area on the test
subjects' face was significantly reduced.
The water insoluble fraction of Astragalus membranaceus may be
obtained by any standard or known methods or technologies well
known to or commonly used by those skilled in the art. For
instance, the water insoluble fraction of Astragalus membranaceus
may be obtained by the steps of providing Astragalus membranaceus,
preferably in a powder form, extracting it with alcohol to obtain
an alcohol extract, and further extracting the alcohol extract with
water to take a water insoluble fraction. In one preferred
embodiment, the alcohol extract is extracted with an alcohol (such
as 95% alcohol) to obtain an alcohol fraction. However, it should
be noted that the alcohol extract is not limited to the water
insoluble fraction and alcohol fraction of Astragalus membranaceus
described above.
The present invention also provides a cosmetic cream comprising the
water insoluble fraction of Astragalus membranaceus, or an
astragaloside compound according to the invention. And the cosmetic
cream may be formulated with other ingredients for making the basic
cream. For example, the ingredients include but are not limited to
Water, Isononyl Isononanoate, Butylene Glycol, Triethylhexanoin,
Behenyl Alcohol, PEG-100 Hydrogenated Castor Oil, Glyceryl Stearate
SE, Dimethicone, Macadamia Nut Oil, Jojoba Oil, Hydrogenated
Lecithin, Methylparaben, DL-.alpha.-Tocopheryl Acetate,
Butylparaben, Phenoxyethanol and Fragrance.
It is noted that the present invention is not limited to only
applications in cosmetics, other pharmaceutical applications, such
as treatments in diseases associated with a deficiency in collagen
I, collagen III, glucosamine, proline or hyaluronic acid (HA) and
an increased expression or activation of matrix metalloproteinases
(MMPs) in the patients may also be encompassed by the scope of the
invention.
The present invention is more specifically explained by the
following examples. However, it should be noted that the present
invention is not limited to these examples in any manner.
EXAMPLE 1
Preparation of the Water Insoluble Fraction of Astragalus
Membranaceus
Powdered of the roots of Astragalus membranaceus (1.0 kg), from
Formosa Kingstone Bioproduct International Co. Ltd., were extracted
with 95% alcohol (.times.3) at 50.degree. C. for 2 hr. The combined
extract was concentrated in vacuo to yield an alcohol extract about
127.8 g. The alcohol extract (127.8 g) was washed with distilled
water for 3 times to yield a water-insoluble fraction (33.9 g) and
a water-soluble fraction (93.9 g).--The alcohol extract (100.0 g)
was dissolved in distilled water and partitioned (1:1) with
n-butanol, and then a butanol fraction (63.0 g) was obtained.
EXAMPLE 2
Effects of the Water Insoluble Fraction of Astragalus Membranaceus
and Astragalosides on Collagen Expression in Human HaCaT Cells and
Human HDF Cells
HaCaT Cell Culture
The Spontaneously transformed human keratinocyte cell lines HaCaT
cells were grown at 37.degree. C. in a humidified incubator under
5% CO.sub.2 and 95% air in culture medium such as Dulbecco Modified
Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS),
100 IU/mL penicillin, 100 .mu.g/mL of streptomycin, 2 mM sodium
pyruvate and 1% Non-Essential Amino Acids (NEAA). The culture
medium was replaced every other day. After confluence, the cells
were subcultured following trypsinization. For subcultures cells
were harvested after brief treatment with 0.1%
trypsin/ethylenediaminetetraacetic acid (EDTA) solution and seeded
at a dilution of 1:10. Cells between passages 12 and 45 were used
for studies. The cells were seeded and incubated for 24 h before
treated with the astragalosides for 24 h. Control cultures were
maintained in medium supplemented with vehicle (dimethyl sulfoxide
(DMSO) 0.1%). No growth and differentiation effects of DMSO were
observed under these culture conditions.
HDF Cell Culture
Primary human dermal fibroblasts cells (HDF) were purchased from
Cascade Biologics (Portland, Oreg., USA) and cultured in medium 106
supplemented with 10% fetal bovine serum (FBS), 100 IU/mL
penicillin, 100 .mu.g/mL of streptomycin. The cells from passages 4
to 10 were used for this study. Culture medium was replaced every
other day. After confluence, the cells were subcultured following
trypsinization. For subcultures cells were harvested after brief
treatment with 0.1% trypsin/EDTA solution and seeded at a dilution
of 1:10. The cells were seeded at 1.times.10.sup.5 cells in 6-well
culture plate for 24 h before treated with astragalosides or crude
extracts, such as butanol fraction and water insoluble fraction of
Astragalus membranaceus for the indicated time periods. Control
cultures were maintained in medium supplemented with vehicle
(dimethyl sulfoxide, DMSO, 0.1%). No growth and differentiation
effects of DMSO were observed under these culture conditions.
Astragaloside Treatment
The cells were treated with pure compounds such as astragaloside I
(AS1), astragaloside II (AS2), astragaloside III (AS3),
astragaloside IV (AS4), astragaloside VI (AS6), isoastragaloside I
(isoAS1), isoastragaloside II (isoAS2), and cycloastragenol
6-O-.beta.-D-glucopyranose (AA) at concentrations of 0-10 .mu.M.
Alternatively, the cells were also treated with the crude extracts,
such as butanol fraction and water insoluble fraction of Astragalus
membranaceus at concentrations of 0, 1, 10, and 40 .mu.g/mL. The
cells were treated with the indicated compounds or the extracts for
24 h before harvested for analysis of collagen I and III expressed
in the whole cell extract or secreted into the culture medium.
Western Blot
Western blot analysis was carried out on both of cultured medium
and cell lysates. The cells were plated on 6-cm dishes at a density
of 1.times.10.sup.6 cells/dish for 24 h prior to treatment with
indicated concentrations of astragalus triterpenoids as indicated
for another 48 h. The conditioned culture mediums were then
centrifuged, 2-fold concentrated and collected for assay of
collagens and MMPs secreted into the culture medium. The cells were
washed and lysed in 0.2 mL of lysis buffer (1% NP-40, 50 mM
Tris-HCl, pH 7.4, 180 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM NaF, 10
mM Na.sub.3VO.sub.4) for 30 min at 4.degree. C. After centrifuging
at 17500 g for 15 min, the supernatants of cell lysate were also
collected. Protein concentration of the samples were measured using
the bicinchoninic acid (BCA) protein assay kit according to the
manufacturer's protocol (Pierce, Rockford, Ill., USA). Equal amount
of protein samples of concentrated conditioned medium (100 .mu.g)
or cell lysate supernatants (50 .mu.g) were mixed with an
appropriate volume of 4.times.SDS sampling buffer and separated by
8% SDS-PAGE gel. The protein bands separated in the SDS-PAGE gel
were blotted onto a polyvinylidene fluoride (PVDF) membrane. The
blotted PVDF membrane was washed twice and blocked in freshly
prepared Tris-buffered saline (TBS) containing 0.1% Tween-20 and 7%
skim milk (TBST) for 2 h at room temperature. The PVDF membrane was
then incubated with either antibodies against collagen I and III or
the housekeeping protein .alpha.-tubulin (Santa Cruz Biotechnology,
Santa Cruz, Calif.) for 18 h at 4.degree. C. Horseradish
peroxidase-conjugated anti-goat antibody was used as secondary
antibody. Signals were visualized by an enhanced chemiluminescence
kit (Clonetech, Palo Alto, Calif., USA) followed by exposure to
X-ray films.
Results shown represent the quantitated data from western blot
analysis. Generally, as shown in Table 1A, the collagen I was
over-expressed in the human HaCaT cells treated with different
dosages (0.01, 0.1 and 1 .mu.M) of the astragalosides, such as AS1,
AS2, isoAS1, isoAS2 and AA. The collagen I was also over-expressed
in the human HaCaT cells treated with 40 .mu.g/mL of astragaloside
extract.
TABLE-US-00001 TABLE 1A Collagen I in cell compound (in .mu.M) 0
0.01 0.1 1 AS1 1.00 1.13 0.72 1.22 AS2 1.00 0.86 1.14 1.87 isoAS1
1.00 1.13 1.06 1.19 isoAS2 1.00 1.52 0.94 1.41 AA 1.00 2.57 3.34
3.47 Collagen 1 in cell compound (in .mu.g/mL) 0 1 10 40 water
insoluble fraction 1.00 1.00 0.95 1.13
The collagen III was also over-expressed in the human HaCaT cells
treated with different doses (0.01, 0.1 and 1 .mu.M) of the
astragalosides, such as AS1, AS2 and AS3. Similarly, the collagen
III was over-expressed in the human HaCaT cells treated with
various dosages (1, 10 and 40 .mu.g/mL) of the crude extract as
shown in Table 1B. On the other hand, the amount of collagen III
secreted into the medium was measured to determine the effect on
cellular release of collagen III. In table 1B, the collagen III was
over-expressed in the cell medium when the human HaCaT cells were
treated with different dosages (0.01, 0.1 and 1 .mu.M) of the
astragalosides, such as AS4, AS6 and isoAS1.
TABLE-US-00002 TABLE 1B Collagen III in cell compound (in .mu.M) 0
0.01 0.1 1 AS1 1.00 1.29 1.59 2.63 AS2 1.00 1.36 1.44 1.49 AS3 1.00
0.86 1.22 2.12 Collagen III in cell compound (in .mu.g/mL) 0 1 10
40 water insoluble fraction 1.00 1.28 2.66 1.88 Collagen III in
medium compound (in .mu.M) 0 0.01 0.1 1 AS4 1.00 0.79 1.13 1.16 AS6
1.00 4.55 4.27 3.86 isoAS1 1.00 3.32 4.41 6.53
The collagen expression in the human HDF cells was also determined.
According to the results listed in Table 2A, it was found that
collagen I was over-expressed in the human HDF cells treated with
the astragalosides including AS2, AS4, AS6, isoAS1 and AA. And
Collage I was over-expressed in the cells treated with various
doses (1, 10, 40 .mu.g/mL) of the crude extracts, such as butanol
fraction and water insoluble fraction of Astragalus membranaceus.
In addition, the expression of collagen I was also observed in the
culture medium, suggesting that an increased amount of the collagen
I was secreted into the medium when the cells were treated with
either the astragalosides or the crude extract.
TABLE-US-00003 TABLE 2A compound Collagen I in cell Collagen I in
medium (in .mu.M) 0 0.01 0.1 1 0 0.01 0.1 1 AS2 1.00 1.02 0.73 1.26
1.00 1.24 1.15 1.02 AS4 1.00 1.16 1.33 1.07 1.00 1.24 1.11 1.11 AS6
1.00 1.18 1.41 1.30 1.00 1.19 1.29 1.25 isoAS1 1.00 0.71 1.11 1.27
1.00 1.20 1.08 0.96 AA 1.00 1.04 1.85 1.17 1.00 0.97 0.95 1.52
compound Collagen I in cell Collagen I in medium (in .mu.g/mL) 0 1
10 40 0 1 10 40 butanol fraction 1.00 1.88 1.62 2.29 1.00 1.12 1.08
1.20 water insoluble 1.00 0.95 0.99 1.63 1.00 1.28 1.40 1.72
fraction
Moreover, as shown in Table 2B below, the expression of collagen
III was also observed both in the cell and in the culture medium
when the human HDF cells were treated with different doses of the
crude extracts. Therefore, the results from the western blotting
analysis generally suggest over-expression of the collagen I and
collagen III in human HaCaT and HDF cells and their increased
release into the culture medium.
TABLE-US-00004 TABLE 2B Collagen III in cell compound (in .mu.M) 0
0.01 0.1 1 AS1 1.00 1.16 1.08 1.18 AS6 1.00 1.65 1.37 0.95 compound
(in .mu.g/mL) 0 1 10 40 butanol fraction 1.00 0.69 1.45 2.75 water
insoluble fraction 1.00 2.71 3.10 1.81 Collagen III in medium
compound (in .mu.M) 0 0.01 0.1 1 AS1 1.00 1.38 1.19 1.77 AS2 1.00
1.26 1.74 0.94 AS3 1.00 2.18 1.79 1.51 isoAS2 1.00 1.00 1.07 1.18
AA 1.00 1.03 1.62 1.34 compound (in .mu.g/mL) 0 1 10 40 water
insoluble fraction 1.00 1.55 2.32 2.83
EXAMPLE 3
Effect of Astragalosides on Proline Uptake in Human HaCaT Cells
Proline Uptake Assay
The proline uptake assay was carried out as described (Biochim
Biophys Acta. 1104:283-292, 1992). Briefly, HaCaT cells were seeded
into a 24-well plate at a density of 3.times.10.sup.4 cells/well
and cultured for 24 h. The cells were then treated in the absence
(solvent control) or presence of various concentrations of
astragalosides or crude extracts from Astragalus membranaceus for
another 48 h. The treated cells were then washed once with PBS and
incubated in amino acid free medium (AAFM) for another 30 min. The
treated cells were them replaced with fresh AAFM containing 50
.mu.g/ml of ascorbate and total of 0.5 mM of L-proline containing 1
.mu.Ci [.sup.3H] Proline (American Radiolabelled Chemicals Inc,
ARC, St. Louis, Mo., USA). At designated time interval, the cells
were washed with AAFM containing cold proline and then lysed in 200
.mu.L of 2% SDS. Cell lysates were centrifuged at 15000 g for 15
min. Intracellular proline uptaken by the cells was determined by
transferring 10 .mu.L of the cell lysate to filter-bottomed
UniFilter plates (Perkim-Elmer) and counted. Protein concentration
of the samples were measured using the BCA protein assay kit as
described above. Proline accumulated in the cells was calculated
and normalized to protein concentration and uptake rate was
expressed as nmole of L-proline per minute per milligram of cell
protein (nmole/min/.mu.g).
TABLE-US-00005 TABLE 3 Uptake rate(pMol/mg/ min) Percentage (%)
Control 0.6904 .+-. 0.0225 100.00 0.1 .mu.M AS1 0.8627 .+-. 0.0741
124.97 0.1 .mu.M AS2 0.9770 .+-. 0.1119 141.52 0.1 .mu.M AS3 0.7213
.+-. 0.0090 104.48 0.1 .mu.M AS4 0.7628 .+-. 0.1015 110.50 2.5
.mu.g/mL water 0.7196 .+-. 0.0308 103.68 insoluble fraction 0.25
.mu.g/mL butanol 0.7288 .+-. 0.0401 105.56 fraction 0.50 .mu.g/mL
butanol 0.8230 .+-. 0.0573 119.20 fraction 1 .mu.g/mL butanol
0.7499 .+-. 0.0577 108.62 fraction
From the results listed in Table 3, it was found that the crude
extract has enhanced the proline uptake by 124.97%, 119.2% and
108.62% in human HaCaT cells as compared to the control group,
suggesting that the crude extract had a promoting effect on the
cellular uptake of proline.
EXAMPLE 4
Effect of Astragalosides on Hyaluronic Acid (HA) Expression in
Human HaCaT Cells HA Measurement
HA measurement was carried out as described (BBRC, 2004, 316:
348-355). Briefly, HaCaT or HDF cells were seeded and incubated in
24-well plates to confluency. Immediately before experiments, cells
were washed twice with serum-free medium to completely remove HA
accumulated during cell growth. The cells were then cultured with
or without different compounds in 0.5 ml serum-free medium for 48
h. At the indicated time, aliquots of medium were removed,
centrifuged at 15000 g for 5 min, and supernatants were analyzed
for HA using an enzyme-linked immunosorbent assay (ELISA) kit
(Echelon Bioscience, Salt Lake, Utah).
Results shown represent the fold of induction of the astragalosides
on hyaluronic acid expression in HaCaT cells. The concentrations of
astragalosides used were 0, 1, or 10 .mu.M (unless indicated
otherwise) for astragaloside pure compounds; were 0, 0.5, 1, 2.5,
5, 10, 40 .mu.g/mL for the butanol fraction (unless indicated
otherwise); and were 0, 1, 10, 40 .mu.g/mL for the water insoluble
fraction (unless indicated otherwise). The cells were treated with
the indicated compounds for 24 h before harvested for analysis of
secreted hyaluronic acid in culture medium using HA-ELISA kit.
The cells treated with 1 .mu.M or 10 .mu.M of the astragalosides
AS1, AS2, AS4 and AS6 have all shown increased cellular secretion
of HA as evident in the increased HA expression in the medium. And
in the human HaCaT cells, the crude extracts of different doses (1,
10 and 40 .mu.g/mL) have also promoted the cellular secretion of HA
into the medium as shown in Table 4A.
TABLE-US-00006 TABLE 4A HA in medium compound (in .mu.M) 0 1 10 AS1
1.00 1.45 1.36 AS2 1.00 1.98 1.31 AS4 1.00 2.27 1.31 AS6 1.00 1.13
1.38 HA in medium compound (in .mu.g/mL) 0 0.5 1 2.5 5 10 40
butanol fraction 1.00 0.98 1.02 1.02 0.98 1.03 1.33 compound (in
.mu.g/mL) 0 1 10 40 water insoluble 1.00 1.16 1.20 1.19
fraction
Quantitative Analyses of the HA Synthase 2 (HAS2) Transcripts
Relative levels of HAS2 expression in HaCaT cells were determined
by real-time quantitative reverse transcriptase polymerase chain
reaction (qRT-PCR). Total RNAs were isolated from the cultured
human cells using TRIzol reagent (Invitrogen, Irvine, Calif., USA).
1 .mu.g of RNA was reverse transcribed, at 37.degree. C. for 60 min
in a 20 .mu.l transcription mixture containing dNTP (0.5 mM),
oligo-dT (0.1 .mu.g), RNasin (10 units), 1.times.PCR buffer (20 mM
Tris-HCl, pH 8.3; 2.5 mM MgCl.sub.2; 50 mM KCl), and 100 units of
Moloney murine leukemia virus reverse transcriptase (Invitrogen).
The cDNA product was phenol extracted, ethanol precipitated, and
resuspended in 20 .mu.l TE (20 mM Tris-Cl, pH 8.0 and 1 mM EDTA).
qRT-PCR was performed using the Applied Biosystem 7300 system and
pre-developed Taqman Gene Expression Assays (Applied Biosystems,
Foster City, Calif., USA). The reaction mixtures (20-.mu.l total
volume) contained 2 .mu.l of serially diluted cDNA, 10 .mu.l of
Taqman Universal PCR Master Mix (Applied Biosystems), and 1 .mu.l
of either human HAS-2 gene (assay ID Hs 00193435_ml, Applied
Biosystems), or GAPDH primer mix (assay ID Hs99999905_ml, Applied
Biosystems). The reactions were amplified as follows: 50.degree. C.
for 2 min and 95.degree. C. for 10 min, followed by 40 cycles of
95.degree. C. for 1 min and 65.degree. C. for 1 min. Two
independent triplicate experiments were performed for the selected
genes. Relative quantitation was performed using the 7300 software
(Applied Biosystems, Foster City, Calif., USA). Expression of HAS-2
was normalized to GAPDH expression in each sample.
As evident in table 4B, HAS-2 was over-expressed in the human HaCaT
cells regardless of whether the cells were treated with 0.1 .mu.M
of the astragalosides, such as AS2, AS4, isoAS1 or isoAS2 or
treated with 2.5 .mu.g/mL, 5.0 .mu.g/mL of the butanol fraction of
Astragalus membranaceus or 40 .mu.g/mL of the water insoluble
fraction of Astragalus membranaceus. Therefore, the results in
tables 4A and 4B suggest that the astragalosides or the crude
extracts had a promoting effect on the HA expression in the human
HaCaT cells.
TABLE-US-00007 TABLE 4B HAS-2 in cell compound (in .mu.M) 0 0.1 AS2
1.00 2.73 AS4 1.00 1.41 isoAS1 1.00 1.12 isoAS2 1.00 1.28 HAS-2 in
cell compound (in .mu.g/mL) 0 1.0 2.5 5.0 butanol fraction 1.0 0.81
1.40 1.89 compound (in .mu.g/mL) 0 1 10 40 water insoluble fraction
1.00 0.90 0.92 3.63
EXAMPLE 5
Effect of Astragalosides on Glucosamine Uptake in Human HaCaT
Cells
Glucosamine Uptake Assay
Glucosamine is the primary building block of HA. Therefore, a
measurement of glucosamine uptake by the cells suggests synthesis
of HA in human cells.
In glucosamine uptake test, HaCaT cells were seeded into 24-well
plate at a density of 3.times.10.sup.4 cells/well and cultured for
24 h. The cells were then treated in the absence (solvent control)
or presence of various concentrations of astragalosides for another
48 h. The treated cells were then washed twice with PBS and
incubated in glucose and serum free medium (GSFM). After 2 h, the
cells were replaced with fresh GSFM containing 0.2 .mu.Ci of
[.sup.14C]-Glucosamine (American Radiolabelled Chemicals Inc, ARC,
St. Louis, Mo., USA). At the designated time interval, the cells
were washed twice with GSFM containing cold glucosamine and then
lysed in 200 .mu.L 2% SDS. Cell lysates were centrifuged at 15000 g
for 15 min. Intracellular uptaken glucosamine was determined by
transferring 10 .mu.L of the cell lysate to filter-bottemed
UniFilter plates (Perkim-Elmer) and counted. Protein concentration
of the samples was measured using the BCA protein assay kit as
described above. Glucosamine accumulated in the cells was
calculated and normalized to protein concentration and uptake rate
was expressed as counts per minute per microgram of cell protein
(cpm/min/.mu.g).
As shown in Table 5, the glucosamine uptake rate increased when the
cells were treated with 0.1 .mu.M of the astragalosides AS1, AS2,
AS3, IsoAS1 and IsoAS2, indicating promoting effect of the
astragaloside on the glucosamine uptake.
TABLE-US-00008 TABLE 5 Uptake rate(CPM/.mu.g protein) Percentage
(%) Control 0.8242 .+-. 0.0321 0.1 .mu.M AS1 1.0482 .+-. 0.0560
127.18 0.1 .mu.M AS2 0.9561 .+-. 0.0918 116.00 0.1 .mu.M AS3 1.0129
.+-. 0.0371 122.90 0.1 .mu.M AS4 0.8931 .+-. 0.0918 108.36 0.1
.mu.M AS6 0.8471 .+-. 0.0278 102.78 0.1 .mu.M AA 0.9445 .+-. 0.0823
114.59 0.1 .mu.M IsoAS1 0.9589 .+-. 0.0665 116.34 0.1 .mu.M IsoAS2
0.9226 .+-. 0.0668 111.94
EXAMPLE 6
Effect of Astragalosides on Matrix Metalloproteinase (MMPs)
Expression in Human HaCaT-1 and HDF-1 Cells
Matrix metalloproteinase-1 (MMP-1) is an enzyme that digests
interstitial collagens and other extracellular matrix
macromolecules. Matrix metalloproteinase-1 is the primary
collagenase Therefore, a presence of the MMP-1 or its proteolytic
activity would indicate that the collagen is down-regulated. On the
other hand, a reduced activity or expression of the MMP-1 would
suggest that the collagen may be abundant in the cells.
UV Irradiation
To characterize the effects of astragalosides on the expression
level of MMPs in human cells, HDF or HaCaT cells were first treated
with or without various concentrations of astragalosides for 24 h.
The cells were washed with phosphate-buffered saline (PBS) and
irradiated with 10-100 mJ/cm.sup.2 of UV-B (312 nm) for HDF-1 and
HaCaT cells, respectively, using the UV light irradiator (UVItec
unlimited, Cambridge, England). The cells were then washed and
incubated with serum free medium for 24 h before harvested for
analysis. The effect of astragalosides on MMPs levels was analyzed
by western blot and the activity of MMPs was analyzed by
zymography. All trans retinoic acid (atRA) was used as a positive
control to suppress the UV-B-induced MMPs activation and
expression.
Human HDF cells were treated with the indicated concentrations of
water insoluble fraction of Astragalus membranaceus for 24 h. The
cells were then irradiated with 50 mJ/cm.sup.2 of UV-B. After 24 h,
the cells were harvested for western blot analysis. As shown in
FIG. 1, a basal level of MMP-1 was expressed in cells not
irradiated with UV (lane 1, control), UV irradiation induced MMP-1
expression in HDF cells (lane 2), all-trans R.sub.A (atRA) blocked
the UV-mediated induction of MMP-1 expression (lane 3, positive
control), and the UV-induced MMP-1 expressions were inhibited in
cells pre-treated with 0.01, 1, 10, and 40 mM of water insoluble
fraction of Astragalus membranaceus (lane 4 to 7). The results
demonstrated that the water insoluble fraction of Astragalus
membranaceus was efficient in blocking MMP-1 expression triggered
by UV irradiation, which suggested that this compound could protect
collagen I from degradation elicited by MMP-1. Thus, the water
insoluble fraction of Astragalus membranaceus is a potential agent
for the prevention and treatment of skin aging as a result of UV
irradiation.
Zymography
The proteolytic activities of MMPs from HaCaT and HDF cells were
measured essentially as described elsewhere (Circ Res, 1999;
85:906-911). The cells were seeded for 24 h and then treated with
various concentrations of astragalosides for another 48 h. The
conditioned culture medium was then centrifuged, 2-fold
concentrated, and saved for assay of collagens and MMPs expressed
into the culture medium. The cells were then lysed in lysis buffer
(1% Triton, 50 mM Tris-Cl, pH 7.4, 180 mM NaCl, 1 mM EDTA). Protein
concentration of the samples was measured using the bicinchoninic
acid (BCA) protein assay kit as described. Samples of concentrated
conditioned medium and cell lysates were mixed with non-reducing
electrophoresis loading buffer and subjected to electrophoresis
under non-reducing conditions, which was carried out on a 10%
SDS-PAGE co-polymerized with 2 mg/mL gelatin or casein (Sigma, St.
Louis, Mo. USA). For zymographic analysis of pro-MMP-2 and
activated MMP-2, the total 20 .mu.g of culture medium were used for
electrophoresis. Following electrophoresis, renaturation of the
proteins was achieved by incubating the gels in 25 g/L Triton X-100
at room temperature for 2.times.10 min. Subsequently the gels were
incubated in 50 mM Tris-HCl, pH 7.5 containing 0.2 M NaCl, 0.02%
Brij35 and 10 mM CaCl.sub.2 at 37.degree. C. for 18 h. After
staining the gels with Coomassie Brilliant Blue R-250 and
destaining, zones of proteolytic activities became visible as
transparent bands in the stained gel. In order to obtain higher
sensitivity, the gels were further destained in a solution of 1%
Triton x-100 (1 to 2 h). This procedure increased the
signal-to-noise ratio, allowing the visualization of faint
gelatinase bands at 92, 125 and greater than 200 kDa. Zymograms
were read using a Molecular Dynamics (Sunnyvale, Calif., USA)
computing laser densitometer with Image Quant software.
Quantitative Analyses of the MMP-1 Transcripts
Relative levels of MMP-1 expression in HDF cells were determined by
qRT-PCR. Preparation of total RNA and cDNA were carried out as
described in previous sections. qRT-PCR was performed using the
Applied Biosystem 7300 system and pre-developed Taqman Gene
Expression Assays (Applied Biosystems, Foster City, Calif., USA).
The reaction was performed as described in previous sections except
that primer mix for human MMP-1 (assay ID HS00233958_ml, Applied
Biosystems) and GAPDH (assay ID Hs99999905_ml, Applied Biosystems)
were used.
Results shown in Tables 6A and 6B represent the quantitated data
from zymography analysis of MMP-2 protein in HaCaT cells and
qRT-PCR for MMP-1 in HDF cells. The concentrations used were 0,
0.01. 0.1, 1 .mu.M (if not indicated otherwise) for pure compounds;
and were 0, 1, 10, 40 .mu.g/mL for crude extract (if not indicated
otherwise). The cells were treated with the indicated compounds for
24 h before harvested for analysis of Pro-MMP-2 or activated MMP-2
in whole cell extract. Fifty micrograms of the cell extract samples
were used for electrophoresis.
In accordance with the results in Table 6A, the proteolytic
activity of the MMP-2 in the conditioned medium was reduced or
weakened when the cells were treated with various doses (0.01, 0.1
and 1 .mu.M) of the astragalosides AS1, AS2, AS3 and AS6,
suggesting the collagen was abundant in the human HaCaT cells.
TABLE-US-00009 TABLE 6A ProMMP-2/MMP2 in medium compound (in .mu.M)
0 0.01 0.1 1 AS1 1.00 0.57 0.57 0.86 AS2 1.00 0.89 0.89 0.67 AS3
1.00 0.76 0.57 0.81 AS6 1.00 0.89 0.67 0.74
The expression of MMP-1 mRNA was reduced in the HDF cells treated
with 0.1 .mu.M of the astragalosides AS1, AS2, isoAS1, isoAS2 and
AA as shown in Table 6B. In addition, the expression of MMP-1 mRNA
decreased when the cells were treated with various doses (1 and 10
.mu.g/mL) of the crude extracts. Therefore, the results from the
tables 6A and 6B both suggest that an effective amount of the
astragalosides or crude extracts would reduce the expression of
MMP-1 and suppress collagen degradation elicited by this
enzyme.
TABLE-US-00010 TABLE 6B MMP-1 Q-PCR compound (in .mu.M) 0 0.1 AS1
1.00 0.75 AS2 1.00 0.25 isoAS1 1.00 0.6 isoAS2 1.00 0.71 AA 1.00
0.76 MMP-1 Q-PCR compound (in .mu.g/mL) 0 1.0 2.5 5.0 butanol
fraction 1.0 0.64 0.77 0.67 compound (in .mu.g/mL) 0 1 10 water
insoluble fraction 1.00 0.44 0.34
EXAMPLE 7
Preparation of Cosmetic Cream Containing Crude Extract of
Astragalus Membranaceus
The cosmetic cream was prepared by mixing components for making the
basic cream with an effective amount of the astragaloside extract
serving as an active ingredient of the cosmetic cream. The
components included but not limited to various amounts of water,
Isononyl Isononanoate, Butylene Glycol, Triethylhexanoin, Behenyl
Alcohol, PEG-100 Hydrogenated Castor Oil, Glyceryl Stearate SE,
Dimethicone, Macadamia Nut Oil, Jojoba Oil, Hydrogenated Lecithin,
Methylparaben, DL-.alpha.-Tocopheryl Acetate, Butylparaben,
Phenoxyethanol and Fragrance as listed in the Table 7.
TABLE-US-00011 TABLE 7 Cosmetic Cream containing H.sub.2O insoluble
fraction of extract from Astragalus Basic Cream (in Components
membranaceus (in 100 g) 100 g) Water to 100.00 g (or %) to 100.00 g
Isononyl Isononanoate 7.00 g 7.00 g Butylene Glycol 5.00 g 5.00 g
Triethylhexanoin 3.00 g 3.00 g Behenyl Alcohol 3.00 g 3.00 g
PEG-100 Hydrogenated 2.00 g 2.00 g Castor Oil Glyceryl Stearate SE
1.00 g 1.00 g Dimethicone 1.00 g 1.00 g Macadamia Nut Oil 0.50 g
0.50 g Jojoba Oil 0.50 g 0.50 g Hydrogenated Lecithin 0.20 g 0.20 g
Methylparaben 0.15 g 0.15 g DL-.alpha.-Tocopheryl Acetate 0.10 g
0.10 g Butylparaben 0.10 g 0.10 g Phenoxyethanol 0.10 g 0.10 g
Fragrance 0.07 g 0.07 g Water insoluble fraction 0.02 g --
EXAMPLE 8
Clinical Trial
Test Subject
Twenty-five healthy volunteers (20 females and 5 males) aged from
35 years old and above were admitted to the clinical study with
their consents. The test subjects were asked not to put any other
cosmetic products on their faces during the whole period of study.
The physical properties (skin pH, skin moisture content,
transepidermal water loss, skin surface lipid, skin color and
brightness, skin elasticity and skin roughness) of the skin were
evaluated 30 minutes after the cleansing the subjects' faces to
provide baseline measurements. Next, a suitable amount (e.g. size
of a peanut) of the cosmetic cream comprising the extract of the
invention was applied evenly on right side of the subject's face
and equal amount of the basic cream was applied on the left side of
the face to provide a comparison, and the same measurements were
taken again 30 minutes after the creams were applied on the
subject's face to provide short term effect data. All the tests
were conducted at a room temperature ranged from about 24.degree.
C. to about 28.degree. C., a relative humidity ranged from about
50% to about 60%. Each of the tests was carried out with each
measurement taken from a test spot having a diameter of about 2 cm
at three selected test areas (forehead, corner of the eye and upper
cheek) on either side of the test subject's face.
Skin pH:
The measurements were made using Skin-pHMeter.RTM. PH 905
(Courage+Khazaka electronic) which had a probe equipped with sensor
elements. The planar design of the probe head allowed direct
measurement with skin contact.
Skin Color and Brightness (Ervthematic Index & Melanin
Index):
The measurement was based on absorption/reflection of the light.
The probe of the Mexameter.RTM. MX 18 (Courage+Khazaka electronic)
emitted three specific light wavelengths. A receiver measured the
light reflected by the skin. The positions of emitter and receiver
ensured that only diffused and scattered light was measured. As the
quantity of emitted light was defined, the quantity of light
absorbed by the skin could be calculated. The melanin was measured
by specific wavelengths chosen to correspond to different
absorption rates by the pigments. The specific wavelengths
corresponding to the spectral absorption peak of haemoglobin were
also selected for the erythema measurement to avoid other color
influences (e.g. bilirubin). The results for both parameters were
shown as indices.
Skin Roughness:
The skin roughness was measured using Skin Visiometer SV
(Courage+Khazaka Electronic, Koln, Germany) based on light
transmission through a thin replica comprising blue-dyed
two-component silicone of which the light absorption was known.
With the thin replica inserted in a modified slide projector, light
coming directly from a neon lamp penetrated the replica and was
absorbed depending on the thickness of the silicone material. A
video sensor charge couple device and a black and white (b/w)
CMOS-camera with a resolution of 640.times.480 pixels measured the
amount of locally transmitted light, and the light intensity was
calculated according to Lambert and Beer's law of absorption.
The averaged measurements of the physical properties taken from 15
test subjects aged from 38 to 58 yrs old after 30 days of the
treatment were listed in Table 8.
TABLE-US-00012 TABLE 8 pH Erythematic Melanin Item value Index
Index L value Surface Volume left side -8.00 -4.25 -22.95 0.68
-2.79 3.29 right side -11.19 0.22 -27.17 -1.18 -4.77 -7.77
In accordance with the results shown in Table 8, a drop of averaged
skin pH by 11.19% was detected on the right side of the test
subject's face. So, the skin would be expected to maintain in a
healthy condition with minimum growth of microorganisms due to
slightly acidic environment achieved when the skin was treated with
the cosmetic cream containing the water insoluble fraction of
Astragalus membranaceus of the invention. In view of an averaged
erythrematic index of 0.22 detected, the right side of the test
subject's face was more erythematic than the left side, suggesting
that the right side of the face had a more efficient blood
circulation. The melanin index measured on the right side of the
face indicated that amount of melanin was reduced by 27.17%. Also,
the skin brightness as measured in terms of L value was evaluated
together with the erythrematic index and the melanin index. A drop
of the L value by 1.18% suggested that the skin looked brighter
after the cosmetic cream was applied for one month. Accordingly,
the right side of the test subject's face would look as a whole
more whitening and radiant than the left side.
The surface area of rufflings or folds was measured to determine
the skin wrinkles on the test subject's face. As shown in Table 8,
the average surface area decreased by 4.77% to reduce the skin
wrinkles after the right side of the test subject's face was
applied with the cosmetic cream for one month. The volume which
measured the volume of the skin relative to the skin surface area
was reduced by 7.77%. As a result, the depth and number of the
wrinkles were reduced.
The compound may be administered to the subject in need of skin
care as frequently as several times daily, or it may be
administered less frequently, such as once a day, once a week, once
every two weeks, once a month. The frequency of the dose will be
readily apparent to the skilled artisan and will depend upon any
number of factors, such as, but not limited to, the type and age of
the subject, etc.
It will be appreciated by those skilled in the art that changes
could be made to the embodiments described above without departing
from the broad inventive concept thereof. It is understood,
therefore, that this invention is not limited to the particular
embodiments disclosed, but it is intended to cover modifications
within the spirit and scope of the present invention as defined by
the appended claims.
* * * * *